酿酒酵母全基因组范围内筛选MTM1基因的抑制基因

MTM1 基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1 基因的缺失会严重影响酵母锰超氧化物歧化酶活性,并损伤线粒体功能,因此在非发酵培养基上不能生长．利用MTM1 基因缺失的突变体在非发酵培养基上的生长缺陷,转入酵母基因组文库筛选MTM1 抑制基因,发现MTM1基因缺失造成的损伤一旦形成不可逆转,重新引入MTM1 基因也无法挽救,直接筛选无法得到抑制基因．为了避免MTM1缺失造成的不可逆损伤,在野生型酵母中先转入带有MTM1 基因的质粒,再敲除染色体上的MTM1 基因,随后转入基因组文库,再利用药物5-氟乳清酸(5-FOA)迫使细胞丢失表达MTM1基因的外源质粒,再筛选能在非发酵培养基上生长的转化子,通过这种方法筛选发现,POR2等5个基因的过表达可以挽救MTM1 基因缺失造成的非发酵培养基上的生长缺陷,为深入了解MTM1基因的功能提供了线索,对筛选其他造成不可逆损伤的突变基因的抑制基因提供了一条可行的研究思路．     

利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP),并转化宿主菌株C. tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm,并转化C. tropicalis XZX03菌株,获得转化子C. tropicalis XZX04。经PCR和DNA测序确认转化子C. tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。     

泡盛曲霉抗FOA突变株的筛选与转化

根据泡盛曲霉SG1菌株分生孢子的紫外线致死曲线,选择死亡率为85%～90%的诱变时间诱变分生孢子,然后将其涂布于含FOA(5-flourooroticacid)和尿嘧啶核苷的基本培养基上,选择抗FOA的突变株。经过纯化和回复突变检测后,获得了5株需要尿嘧啶或尿嘧啶核苷才能在基本培养基上生长的稳定突变株。进一步分析鉴定结果表明,这些突变株的URA3基因发生了突变。Northern杂交及RTPCR方法证明这些突变株中URA3基因突变均发生在转录水平上。选择突变株SA5作为受体菌,用含来自黑曲霉的野生型URA3基因的质粒转化该受体菌,结果获得了稳定的转化子。Southern杂交证明野生型URA3基因取代了突变株的ura3基因。     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍（MNNG）诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6～2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1～2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍(MNNG)诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6~2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1~2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

aiiA基因表达载体的构建及其在毕赤酵母中的表达

构建携带N-酰基高丝氨酸内酯酶基因(aiiA)的重组毕赤酵母表达载体pPIC3.5K-aiiA,采用电转化方法转入毕赤酵母GS115,经营养缺陷型培养基、表型鉴定和高G418浓度筛选获得高拷贝表达盒的酵母转化子,用0.5%甲醇诱导表达,RT-PCR鉴定可检测到重组酵母中编码目的基因成熟肽的mRNA,SDS-PAGE和Western blot检测结果表明,aiiA基因在毕赤酵母中成功表达,用指示菌紫色杆菌CV026检测发现目的蛋白具有降解N-酰基高丝氨酸内酯的活性。     

缺铁应答基因FOX1上游调控基因的筛选

[目的]筛选与莱茵衣藻FOX1基因启动子结合的调控基因序列。[方法]利用酵母单杂交的方法,构建pHIS2.1-FOX1诱饵载体,并将其转入到酵母菌株Y187中,在SD/-Trp/-His/-Leu/50 mmol/L 3-AT筛选培养基上挑选酵母阳性转化子,PCR克隆阳性转化子的序列并测序,利用Blastx程序检索phytozome莱茵衣藻基因组数据库,获得阳性转化子序列的同源基因信息。[结果]18个酵母阳性转化子测序成功,其同源基因主要有CTR型铜离子转运体(CTR2)、核糖体蛋白,和参与光合作用、氧化还原反应和物质运输等方面的功能蛋白。[结论]利用酵母单杂交技术筛选到缺铁应答基因FOX1潜在的上游调控基因。     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究,我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因,并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3力标记基因,在不舍尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株,该菌株在低K 培养基中导入拟南芥K 转运体基因AtKuP1可恢复正常生长.     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究, 我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因, 并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3为标记基因, 在不含尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株, 该菌株在低K+培养基中导入拟南芥K+转运体基因AtKuP1可恢复正常生长。     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Properties of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth

In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.     

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.     

Multiple New Genes That Determine Activity for the First Step of Leucine Biosynthesis in Saccharomyces Cerevisiae

The first step in the biosynthesis of leucine is catalyzed by α-isopropylmalate (α-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of α-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu(+) and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of α-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an α-IPM synthase activity. The results demonstrate that multiple components determine the residual α-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.     

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites

We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.     

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.     

抗盐胡杨Na+/H+逆向转运蛋白基因PeNhaD1的功能

将胡杨Na /H 逆向转运蛋白基因PeNhaD1,分别转入对盐敏感的缺失质膜和缺失液泡膜Na /H 逆向转运蛋白基因的酵母突变菌株ANT3和GX1中。结果表明,在pH6.0、Na 浓度为80mmol/L(固体培养基)或400mmol/L(液体培养基)的条件下,转化具有目的基因的酵母ANT3具有更高的耐盐性,而将目的基因转化到突变株GX1时,却不能提高其耐盐性。实验结果说明PeNhaD1可能是通过编码质膜Na /H 逆向转运蛋白而提高酵母的耐盐性的,推测其在胡杨耐盐机制中的作用可能是提高拒盐性。     

Construction of an effective host-vector system for the yeast Saccharomyces exiguus Yp74L-3.

An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast. To obtain a stable reversionless host strain, we constructed an S. exiguus strain carrying leu2::ScURA3 by disrupting the S. exiguus LEU2 gene with the S. cerevisiae URA3 gene. A vector plasmid unique to S. exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S. exiguus into an Escherichia coli phagemid, pUC119. The vector constructed, pTH119 was able to transform the S. exiguus leu2::ScURA3 strain to Leu+ efficiently. The stability of the vector in the S. exiguus host cells resembled that of a YRp-type vector in S. cerevisiae.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.